CN1212017A - Hybrid proteins which form heterodimers - Google Patents

Hybrid proteins which form heterodimers Download PDF

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CN1212017A
CN1212017A CN97192411A CN97192411A CN1212017A CN 1212017 A CN1212017 A CN 1212017A CN 97192411 A CN97192411 A CN 97192411A CN 97192411 A CN97192411 A CN 97192411A CN 1212017 A CN1212017 A CN 1212017A
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CN1261579C (en
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罗伯特·K·坎佩利
布拉德福德·A·詹姆森
斯考特·C·查普尔
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Applied Research Systems ARS Holding NV
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    • C—CHEMISTRY; METALLURGY
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    • C07C209/00—Preparation of compounds containing amino groups bound to a carbon skeleton
    • C07C209/04—Preparation of compounds containing amino groups bound to a carbon skeleton by substitution of functional groups by amino groups
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/24—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
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    • A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P5/14—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K2319/00—Fusion polypeptide

Abstract

A hybrid protein includes two coexpressed amino acid sequences forming a dimer. Each sequence contains the binding portion of a receptor, such as TBP1 or TBP2, or a ligand, such as IL-6, IFN- beta and TPO, linked to a subunit of a heterodimeric proteinaceous hormone, such as hCG. Each coexpressed sequence contains a corresponding hormone subunit so as to form a heterodimer upon expression. Corresponding DNA molecules, expression vectors and host cells are also disclosed as are pharmaceutical compositions and a method of producing such proteins.

Description

Form the hybrid protein of heterodimer
Affiliated technical field
The present invention relates to a kind ofly contain the aminoacid sequence of two co expression and form a dimeric hybrid protein, each of this two seed amino acids sequence contains:
(a) at least a a kind of homology aggressiveness acceptor (homomeric recedtor) that is selected from, a kind of chain of allos aggressiveness, a kind of part, and their segmental aminoacid sequence; And
(b) a kind of subunit of heterodimer protein hormone or its fragment; Wherein (a) and (b) be direct bonded or by a kind of peptide connector bonded, and in each binary, two subunits (b) are different and can assemble the formation dimeric complexes.Background of invention
Protein-protein interaction is absolutely necessary for the normal physiological function of cell and multi-cell organism.The numerous protein of occurring in nature when with one or more other protein chains compound tense mutually, can show that make new advances or the suitableeest function.This can be by various part one receptors bind the pair cell active regulate and illustrate.Some parts, as tumor necrosis factor alpha (TNF α), TNF β, or hCG (hCG) exist with many subunits complex body.Some these species complexs contain same a kind of subunit of multiple copied.TNF α is the homotrimer (1-4) that is formed by 3 identical subunits with TNF β (hereinafter being referred to as TNF).Other part is made up of non-subunit of the same race.As, hCG is a kind of heterodimer (5-7), the form that acceptor also can the multichain complex body exists or function takes place.As, the acceptor of TNF can transmit a signal (8,9) when assembling the formation dimer.The part of these acceptors promotes the gathering of two receptor chains, thereby a kind of mechanism of receptor activation is provided.For example, the gathering of TNF mediation activates TNF acceptor (10-12).
The adjusting of protein-protein interaction can be the useful mechanism that treatment gets involved in various diseases and the pathology.Can part may be separated with acceptor with the solubility conjugated protein of ligand interaction, thereby weaken the activation of specific receptor pathway.Perhaps, but its removing of separation deferrable or the degraded of part, thus increase between its effector phase, and may strengthen its apparent activity in vivo.For TNF, soluble TNF acceptor mainly and the active inhibitory phase of TNF get in touch (13-17).
The solubility conjugated protein comes in handy for the treatment human diseases.For example, soluble TNF acceptor has demonstrated effectiveness (18,19) in arthritic animal model.
Because TNF has 3 binding sites (10-12) to its acceptor, and the Dimerized of this cell surface receptor is enough (8 for biological activity, 9), very possible single soluble receptors will bring a kind of possibility with combining of TNF, promptly this 1: 3 complex body of solubility acceptor: TNF (tripolymer) still can in conjunction with and the activation a pair of cell surface TNF acceptor.For obtaining a kind of retarding effect, two in the receptor binding site on the expectation TNF tripolymer must be occupied or be sealed by the solubility conjugated protein.Perhaps, this conjugated protein can seal suitably directed TNF on cell surface.
Generally speaking, people need to feel the synthetic protein as the dimer hybrid protein that contains two acceptors (or part) chain.Referring to Wallach etc., United States Patent (USP) 5478925.Be used to produce the strategy that contains from the dimer in the outer receptors bind zone of born of the same parents or polymer hybrid protein for these protein are merged mutually with the constant section of heavy chain of antibody.
This strategy has caused, for example, and the structure of CD4 immunoadhesin (20).Two (or whole four) immunoglobulinses zones, front of the CD4 that they are merged by the constant section with the heavy chain of antibody and light chain are formed.This strategy of creating hybrid molecule is adjusted the acceptor (10,16,21) that is used for TNF, and has caused comparing with monomer solubility conjugated protein the generation of the construct with higher external activity.
People are common to be thought, the higher external activity of dimer fusion protein matter should be converted into higher activity in vivo.The result of a research has supported this argument, and p75 (TBP2)-Ig fused protein avoids producing the protection mouse on the adverse consequences of intravenously lps injection and demonstrates at least 50 times high activity.
But though immunoglobulin fusion proteins is used widely, this strategy has several shortcomings.One is that some immunoglobulin fc region territories participate in immune effector function.These functions may be (22) do not expected in specific treatment situation.
Second restriction produces allos aggressiveness fused protein with expectation, and be relevant as the particular condition of the solubility analogue of allos aggressiveness IL-6 or interferon type acceptor.Though have a lot of methods produce the bi-functional antibody (as, merge by cotransfection or hybridoma), generally the mixture of homodimer of Chan Shenging and heterodimer has damaged synthetic validity (23) greatly.Recently existing several reports have been described the assembling (24-26) of using leucine zipper motif guiding heterodimer.This well as if a promising method that is used for research purpose, but in the employed this nonself or born of the same parents sequence owing to its antigenicity is not suitable for clinically life-time service.Stability after assembling efficiency and the assembling also may be limiting factor.
On the other hand, under the particular case of TNF acceptor, found some of p55 TNF acceptor is modified with signal (27,28) when being beneficial to homodimerization and not having part.Have been found that the kytoplasm section of this receptor, be known as " dead section ", can serve as the motif (28,30) of homodimer.As the surrogate of immunoglobulin (Ig) hybrid protein, can imagine that the extracellular region territory of TNF acceptor and the fusion in the dead zone of its kytoplasm can produce a kind of secretory protein, it can be Dimerized when not having TNF.This fused protein discloses and has required patent protection in International Patent Application WO 95/31544.
Dimeric the 3rd strategy that is used to produce soluble TNF acceptor is that this monomeric protein and polyoxyethylene glycol are carried out chemically crosslinked (31).Summary of the invention
Be used to obtain this proteinic another kind of mode one of for the present invention, it has some important advantages, and this mode is to use the natural heterodimer support of the NIg corresponding to have in the recycle system long half-lift.A preferred example is hCG, and it is that a kind of secretion is good, and high stability is arranged and have the protein (32-33) of long transformation period.Because hCG, has developed plurality of reagents as the outstanding effect of conceived marker in vitro and in vivo this protein is carried out quantitatively and research.In addition, use mutafacient system to carry out extensive studies to hCG, and known this proteinic little disappearance, for example remove five residues of the C-terminal of α subunit, can eliminate its biological activity effectively and keep the ability (34,35) that it forms heterodimer simultaneously.Amino and C-terminal that 30 amino acid whose little insertions of as many as have demonstrated at the α subunit have tolerable (36), and the fusion of the C-terminal of α subunit and β subunit simultaneously also has slight influence (37) to the formation of heterodimer.
Also reported the analogue of a kind of hCG, wherein the C-of immunoglobulin fc region territory and hCG β subunit is terminal merges; But this construct is not secreted, and does not also carry out itself and the experiment (38) linked together of α subunit.
Thereby main purpose of the present invention is the hybrid protein that contains the aminoacid sequence that forms a kind of dimeric two kinds of co expression, and each aminoacid sequence contains:
(a) at least a a kind of homology aggressiveness acceptor, a kind of chain of allos aggressiveness acceptor, a kind of part and their the segmental aminoacid sequence of being selected from; With
(b) a kind of subunit of heterodimer protein hormone or its fragment; Wherein (a) and (b) be direct bonded or by a kind of peptide connector bonded, and in each binary, two subunits (b) are different and can assemble and form a dimer complex body.
According to the present invention, this connector can be digested.
Sequence (a) preferably is selected from: the extracellular region territory of TNF acceptor 1 (55KDa is also referred to as TBP1), and their fragment of ligand binding region is perhaps still contained in the extracellular region territory of TNF acceptor 2 (75KDa is also referred to as TBP2); The extracellular region territory of IL-6 acceptor (being also referred to as gp80 and gp130); The extracellular region territory of IFN α/beta receptor or IFN γ acceptor; Gonadotropin acceptor or its extracellular region territory; Light chain of antibody, or its fragment randomly link with corresponding heavy chain; Heavy chain of antibody, or its fragment randomly link with corresponding light chain; The monoclonal antibody zone; Or ligandin matter, as cytokine, somatomedin or the hormone except that gonadotropin, its specific example comprises IL-6, TFN-β, TPO, or their fragment.
Sequence (b) preferably is selected from hCG, FSH, LH, TSH, statin subunit, or their fragment.
Can use these proteinic modifications, as to the chemistry of protein skeleton or enzyme cutting, or, make the component inactivation of hybrid protein of the present invention the chemistry or the enzyme modification of some amino acid side chains.This also can be to active restriction by using recombinant DNA technology, the encoding sequence that changes hybrid protein is finished in the mode that the activity that directly causes a component is restricted, the chemistry or the enzyme modification that perhaps make this protein be easier to accept are subsequently finished.
Based on (b) bonded aminoacid sequence (a), above-mentioned hybrid protein will produce single function, difunctional or polyfunctional molecule.In each binary, (a) can be connected, or link to each other with the both with the aminoterminal or the carboxyl terminal of (b).
A mono-clonal hybrid protein of the present invention is passable, for example, contains a kind of extracellular region territory of gonadotropin acceptor, it and corresponding acceptor-link to each other in conjunction with one of gonadotropin subunit.According to this embodiment, hybrid protein of the present invention can be a kind of like this molecule, and wherein, for example, fsh receptor extracellular region territory links to each other with FSH, to improve the transformation period in the blood plasma and to improve biological activity.
This preparation is in helping propagation method, for example induced ovulation or in vitro fertilization, can be used to induce the folliculus maturation, and serve as the bioactive instrument of violent amplification, thereby be reduced to amount that reaches ovulation needed hormone itself and the number of times that needs injection to the necessary hormone of success of this process.
The production in fsh receptor and human fsh receptor extracellular region territory is described in WO92/16620 and WO96/38575 respectively.
According to a specific embodiment, the extracellular region territory (ECD) of fsh receptor can be merged (29) with frame and present a kind of " being tacked " long-armed with the peptide connector that contains zymoplasm identification/cleavage site.This peptide connector links to each other the extracellular region territory of FSH with the FSH subunit, like this, the extracellular region territory that can make fsh receptor in the recycle system when this molecule contacts with zymoplasm is by being removed in the cutting of zymoplasm cleavage site.
In another embodiment, use be the enzyme recognition site that is present in the enzyme in the ovary in a large number, rather than zymoplasm recognition site.Like this, when the ECD-FSH molecular migration is in ovary, it will be exposed to the enzyme that this tissue middle and high concentration exists, and ECD will be removed, thereby FSH can be interacted with membrane-bound receptor.
In another embodiment, be not to use enzyme recognition site, but a kind of flexible hinge section is cloned between ECD and the FSH, thereby ECD can not removed from this hormone by enzyme.Like this, after the ECD-FSH molecule arrives at ovary, between the ECD of the fsh receptor that exists on ECD that hinge connects and the gonad cell film competition will appear.
In another preferred embodiment of the present invention, this hybrid protein is made up of the aggregate of a pair of aminoacid sequence, one of them contains TBP1 (or from amino acid 20 to amino acid/11 61 or to some fragments of amino acid/11 90) as (a), α subunit with hCG, as (b), other the β subunit or its fragment that always contain TBP1 (or fragment same as described above) conduct (a) and hCG are as (b).According to this embodiment, based on the specific sequence that is selected as (b) (the full subunit of the β of hCG, or its fragment or modifier), the hybrid albumen that is produced will have a kind of activity (activity of having only TBP1) or several active combination (activity of TNP1 and the activity of hCG).Under latter event, this hybrid protein can be used to, for example, and the metabolism loss in combination therapy Kaposi sarcoma and the acquired immune deficiency syndrome (AIDS) (AIDS).
In another embodiment of the present invention, between two subunits (b), add one or more covalent linkage, to improve the stability of the hybrid protein that is produced.This can be undertaken by for example adding one or more non-natural interchain disulfide bonds.Can derive out in these crosslinked sites from the structure of known heterodimer hormone.For example, replace a salt bridged bond (non covalent bond) for halfcystine can be placed the residue Lys45 of α subunit and the residue Glu21 of β subunit in a suitable site among the hCG with a disulfide linkage (covalent linkage).Another object of the present invention is the PEGization of these hybrid proteins or the form of other chemically modified.
Another purpose of the present invention is a kind of dna molecular, and it contains the dna sequence dna of the above-mentioned hybrid protein of encoding, and substantially the same nucleotide sequence.The nucleotide sequence of given aminoacid sequence that " substantially the same nucleotide sequences " comprises other because the degeneracy of genetic code is also encoded.
For producing hybrid protein of the present invention, dna sequence dna (a) derives from the clone of existence, as (b).The dna sequence dna of the sequence (a) that coding is required links to each other with the dna sequence dna of the required sequence of coding (b).These two kinds of fusion products insertions and connection are advanced suitable plasmid or each is connected into different plasmids; after finishing; expression vector or two kinds of expression vectors are imported appropriate host cell, and host cell is expressed carrier then, produces hybrid protein of the present invention as defined above.
Preparing the proteic preferred method of hybrid of the present invention is by round pcr, and the oligonucleotide of use is to having specific oligonucleotide from the expectation sequence that encoding sequence (a) and clone (b) duplicate.
The expression of any recombinant protein of the present invention as herein described can use suitable expression to carry out in eukaryotic cell (as yeast, insect or mammalian cell) or prokaryotic cell prokaryocyte, can use the known any method of prior art.
For example, the protein DNA molecule that coding is obtained by above-mentioned any one method inserts in the expression vector that suitably makes up (referring to Sambrook etc. 1989) with the prior art technique known.The technology that double-stranded cDNA is connected with plasmid vector has homology polymer tailing, relate to the restricted connection and flush end interconnection technique of using synthetic DNA connector: use dna ligase to be connected dna molecular, and the connection by avoiding with alkaline phosphatase treatment not expecting.
In order to express required protein, an expression vector should also comprise and contains the special nucleotide sequence of transcribing and translating adjusting information, and it links to each other with coding expectation protein DNA, and its mode of connection should allow expression of gene and this proteinic generation.Of paramount importance for the gene that will transcribe is that it must have the promotor that can be discerned by RNA polymerase in its front, thereby this polysaccharase combines initial transcription with promotor.Existing multiple this promotor is come into operation, and they are with different efficient work (strong and weak promoter).
For eucaryon host, can use the different adjusting sequences of transcribing and translate according to host's character.They can be from virus, as adenovirus, and little bovine papilloma virus, simian virus etc., wherein, this conditioning signal links with the specific gene with very high expression level.Their example has the TK promotor of simplexvirus, SV40 early promoter, yeast ga14 gene promoter etc.Selected transcription initiation conditioning signal can be to allow to suppress and activated, thereby can regulate and control the expression of group.
The nucleotide sequence that will contain the hybrid protein of the present invention of encoding inserts in the carrier, this carrier have can handle connection transcribe and translate conditioning signal, and can the expectation gene order be integrated into host cell.The cell of the DNA stable conversion that has been imported into can be selected by also importing one or more markers, and this marker can allow the host cell that contains expression vector is selected.This marker also can provide phototrophy to the auxotroph host, the thing resistance of killing livestock, and as microbiotic, or heavy metal, as copper etc.Selectable marker gene can be directly links to each other with DNA gene order to be expressed, and also can import in the same cell by cotransfection.For the present invention proteinic the most suitable also may needs becoming other key element.
Selecting specific plasmid or the important factor in the virus vector to comprise: the difficulty or ease of discerning and select carrier-containing recipient cell from those the not carrier-containing recipient cell; The copy number of expectation carrier in specific host; And whether need it between different types of host cell, " to shuttle back and forth ".
In case prepare the carrier that contains described construct or the dna sequence dna that are used to express, this DNA construct can be imported into appropriate host cell by in the multiple suitable mode any one: transform transfection, engage, protoplastis merges, electroporation, calcium phosphate precipitation, direct microinjection etc.
Host cell can be prokaryotic cell prokaryocyte or eukaryotic cell.Preferred eukaryotic cell, as mammalian cell, as people, monkey, mouse and Chinese hamster ovary (CHO) cell, this is because they provide protein molecule translates the back and modify, and comprises correct folding and in correct site glycosylation.Yeast cell is also translated the back peptide and is modified, and comprises glycosylation.The recombinant DNA strategy of more existing use strong promoter sequences and high copy number plasmid, it is used in the required protein of production in the yeast.Homing sequence on yeast identification clone's the mammalian genes product, justacrine carries the peptide (being propetide) of homing sequence.
After carrier is introduced, host cell is incubated in the selection substratum, it is selected the growth of carrier-containing cell, and the expression of cloned genes sequence causes the generation of desired protein.
Purifying recombinant proteins is by becoming known for any one method of this purpose, promptly relate to extraction, precipitation, and layer folding, any ordinary method of electrophoresis etc. is carried out.A kind of purifying protein of the present invention that is preferred for is further purified the affine layer folding method of method for use and target protein bonded monoclonal antibody, and this monoclonal antibody produces and is fixed on and is contained on the gel matrix in the post.The unpurified agent flow that will contain this recombinant protein is through this post.This protein will combine with post by specific antibody, and impurity will spill out.After the washing, this protein wash-out is come out by changing change pH values or ionic strength.
The general expression of term as used herein " hybrid protein " contains two or more different protein or its segmental protein.
Term used herein " fused protein " is meant a kind of hybrid protein, and two or more albumen that it is linked to each other by covalency or its fragment are formed.
Term used herein " aggregate " is meant the strong specificity noncovalent interaction of formation between two polypeptide chains, thereby form a complex body, the for example interaction that between the α of heterodimer hormone (as FSH, LH, hCG or TSH) and β subunit, forms.
Term used herein " part " or " ligandin matter " are meant a kind of molecule except that antibody or immunoglobulin (Ig), and it can be by the ligand binding region combination of acceptor; This molecule can be that occurring in nature exists, perhaps can be chemically modified or chemosynthesis.
Term used herein " ligand binding region " is meant a kind of participation part bonded acceptor portion, its normally part or whole basically extracellular region territory.
Term used herein " acceptor " is meant a kind of membranin, and it causes the secondary cellular response with combining of corresponding part, causes the activation or the inhibition of born of the same parents' internal procedure.
On the other hand, the invention provides of the application of this hybrid protein as medicine.This medicine preferably provides with the form of the pharmaceutical composition that contains protein of the present invention and one or more pharmaceutically acceptable carriers and/or vehicle.This pharmaceutical composition has been represented another aspect of the present invention.Brief Description Of Drawings
Consulting accompanying drawing can understand the present invention better, wherein:
Fig. 1 (a) and 1 (b) have represented TBP (20-161)-hCG α and TBP (20-161)-hCG β construct respectively, and corresponding sequence (SEQID NO:1-4).
Fig. 2 (a) and 2 (b) have represented TBP (20-190)-hCG α and TBP (20-190)-hCG β construct respectively, and corresponding sequence (SEQID NO:5-8).
Fig. 3 is the diagram summary of the construct of Fig. 1 and Fig. 2, has represented p55 TNFR1, TBP1 and TBP1 fusion constructs.The connector sequence of last two line displays is SEQ ID NO:9 (ALa-GLy-ALa-ALa-Pro-GLy) and SEQ ID NO:10 (ALa-GLy-GLa-GLy).
Fig. 4 shows the TBP-hCG (20-190) of expressing cho cell to the dose-dependently of TNF α-inductive to the Cytotoxic protective effect of BT-20 cell, and various contrast.
Fig. 5 represents the TBP-hCG (20-190) of COS cell expressing to the dose-dependently of TNF α-inductive to the Cytotoxic protective effect of BT-20, and various contrast.
Fig. 6 shows the TBP-hCG (20-161) of the expressing cho cell of affinity purification to the dose-dependently of TNF α-inductive to the Cytotoxic protective effect of BT-20, and various contrast.Detailed description of the preferred embodiments.
The present invention will be described by embodiment hereinafter, can not be interpreted as limiting by any way the present invention.Embodiment material and method
The clone that is used for this research is to derive from American type culture collection (ATCC), Rockville, and Maryland, except as otherwise noted.CHO-DUKX clone is by the D of MIT, and Houseman derives from the Chasin (39) of Columbia University.The CHO-DUKX cell routine ground that lacks functional dihydrofolate reductase gene is remained in the Eagles substratum (α (+) MEM) that the complete α (+) that replenished 10% calf serum (FBS) improves.The COS-7 cell is remained in the Eagles substratum (DMEM) of the Dulbecco improvement that is supplemented with 10%FBS according to routine.Unless stated otherwise, cell broken up make them remain on logarithmic phase, cultivate reagent and derive from GIBCO (Grand Island, New York).
1, the assembling of the genetic constructs of coding hybrid protein
To the numbering of p55TNF acceptor clone's paper (40), and the numbering of people hCG subunit is based on the numbering (41,42) of Fichdes clone paper based on Wallach.TBP, or this appellation of TNF conjugated protein represent can be in conjunction with the abdomen exterior domain part of the TNF acceptor of TNF.In these embodiments, this DNA construct will be named as TBP one hybrid protein, add partner and the section of indicated TBP in the construct nomenclature.All TBP-hCG constructs all contain human growth hormone (hGH) signal peptide of replacing natural p55 signal sequence.In addition, the location of hGH signal peptide is right after in the front of TBP residue A sp20 it, is desirably in that it becomes first residue in the sophisticated secretory protein.These modify for using hCG optional as this basic thought of partner of this hybrid protein.
(43) that the DNA of this hybrid protein of encoding is to use PCR method to make up.
a．TBP1(20-161)-hCG
Originally TBP-hCG construct is designed to contain the ligand binding region in the extracellular region territory (beginning to comprise residue Cys161 from Asp20) from the p55TNF acceptor, and it is by connector and the hCG α and β subunit (respectively from residue α Cys7 or the β Pro7) fusion of a weak point.This construct hereinafter is called TBP1 (20-161)-hCG, is a kind of two adorned hCG subunits, TBP1 (20-161)-hCG α and TBP1 (20-161)-hCG β, heterodimer.
The Oligonucleolide primers that is used for TBP1 (20-161)-hCG α construct is:
Primer 1 (α β) TTT TCT CGA GAT GGC TAC AGG TAA GCG CCC (SEQ ID NO:11)
Primer 2 (α) ACC TGG GGC ACG ACC GGC ACA GGA GAC ACA CTC GTT TTC
(SEQ?ID?NO:12)
Primer 3 (α) TGT GCC GGT GCT GCC CCA GGT TGC CCA GAA TGC ACG CTA CAG
(SEQ?ID?NO:13)
Primer 4 (α) TTT TGG ATC CTT AAG ATT TGT GAT AAT AAC AAG TAC
(SEQ?ID?NO:14)
Other primer of describing among these primers and these embodiment is to use phosphoramidite chemical process synthetic (ABI, Foster city, Gary Fu Niya) on Applied Biosysrem Model 392 dna synthesizers.
Because two TBP-hCG subunit constructs have identical 5 '-end (being 5 ' of hCH/TBP construct-end), two TBP-hCG subunit constructs all use primer 1 (α β).Other primer that is used for TBP1 (20-161)-hCG β construct is:
Primer 2 (β) CCG TGG ACC AGC ACC AGC ACA GGA GAC ACA CTC GTT TTC
(SEQ?ID?NO:15)
Primer 3 (β) TGT GCT GGT GCT GGT CCA CGG TGC CGC CCC ATC AAT
(SEQ?ID?NO:16)
Primer 4 (β) TTT TGG ATC CTT ATT GTG GGA GGA TCG GGG TG (SEQ ID NO:17)
Primer 2 (α) and 3 (α) are reverse complemental, and cover 3 '-end of p55 extracellular region territory coding section, and 5 '-end of hCG α subunit.Equally, primer 2 (β) and 3 (β) also are reverse complemental, and cover 3 '-end of p55 extracellular region territory coding section and 5 '-end of hCG β subunit.
Each of two TBP-hCG subunit constructs is carried out two PCR reactions.First has used primer 1 (α β) and 2 (α or β), and has used as the coding solubility p55 residue 20-180 of template and have the plasmid (pCMVhGHspcDNA.pA4 plasmid) of hGH signal peptide in its front.Second has been used primer 3 (α or β) and 4 (α or β), and used as the plasmid pSVL-hCG α of template or pSVL-hCG β both one of (44).PCR is to use and derives from New England Biolabs (Beverly, Vent Massachusetts) (TM) polysaccharase, and carrying out according to the method for manufacturer recommendation.25 circulations are carried out in each reaction, and reaction conditions is:
Template DNA 100 μ g
Every kind of primer 1 μ g
Vent (TM) polysaccharase (New Englandl Biolabs) 2U
99 ℃ of sex change 30 seconds,
Annealing: for primer 1 (α β) and 2 (α) be 59 ℃ 30 seconds;
Primer 3 (α) and 4 (α) be 59 ℃ 30 seconds;
Primer 1 (α β) and 2 (β) be 57 ℃ 30 seconds;
Primer 3 (β) and 4 (β) be 63 ℃ 30 seconds;
Extended 75 seconds at 75 ℃.
PCR product electrophoresis on 2% sepharose has been confirmed its size for expectation also with ethidium bromide (ethidiumbromide) dyeing.Then these fragments are carried out purifying by a Wizard post (Promega) and according to the explanation of manufacturers.
Last encoding sequence of TBP1 (20-161)-hCG α is to assemble by the fusion PCR that uses primer 1 (α β) and primer 4 (α), and used from first PCR react obtain from the product of p55 and hCG α fragment purification as template.At first with two templates, they are because overlapping between primer 2 (α) and 3 (α) and the sex change after annealing arrives together carries out 10 round-robin PCR under the situation that does not add any primer.These round-robin conditions condition with previous use basically are identical, and just annealing is carried out under 67 ℃, extends and has carried out 2 minutes.When these 10 loop ends, add primer 1 (α β) and 4 (α), carry out other 10 circulations again.The condition that reaction is taken turns in this back one and previous use identical, be used annealing temperature be 59 ℃, extension has been carried out 75 seconds.
Electrophoresis confirms that to the analytical results of the product of this reaction we have obtained the expectation fragment of about 1100bp on 1% sepharose.With product by a Wizard post with this fragment of purifying, then with its with the digestion of Xba I and BamH I, and on 0.7% low melting-point agarose gel purifying again.The fragment subclone of purifying is advanced plasmid pSVL (pharmacia), this plasmid at first with the digestion of Xba I and BamH I and on 0.8% low melting-point agarose gel purifying again.After connecting with the T4 ligase enzyme, mixture is used to transform the AG1 intestinal bacteria, then it is tiled on the LB/ penbritin flat board 37 ℃ of incubated overnight.The plasmid DNA that derives from the bacterium colony of ammonia benzyl toximycin resistance is analyzed by digesting with Xho and BamH I, to confirm the existence of inset (it is cut) in this digestion.Find that 6 clones contain inset, select one of them (clone 7) to be used for progressive one research and called after pSVLTBPhCG α (containing TBP1 (20-161)-hCG α).Dideoxy dna to inset in this carrier checks order, and (result Ohio) has confirmed that this construct is correct, and does not introduce the variation of non-expectation for use SeguenaseTM, U.S.Biochemicals.Cleveland.
The sequence of final coding TBP1 (20-161)-hCG β is with the fusion PCR assembling of the described identical mode of TBP1 (20-161)-hCG α being used primer 1 (α β) and 4 (β), and has used from the product that derives from p55 that first PCR reacts and hCG β fragment purification as template.The pSVL of the inset that contains expectation that produces is named as pSVLTBPhCG β.
b．TBP(20-190)-hCG
Prepare second group of TBP-hCG protein by TBP (20-161)-hCG construct is modified, contain analogue with generation, to replace the 20-161 section in the primary analogue across the TBP of Asp20 to Thr190.To be undertaken by replacing with the PCR fragment that contains this variation in the fragment between Bgl II among the plasmid pSVLTBPhCG α and the Xba I site, this PCR fragment is to use and merges that PCR produces.These primers are:
Primer 1 TTT TAG ATC TCT TCT TGC ACA GTG GAC (SEQ ID NO:18)
Primer 2 TGT GGT GCC TGA GTC CTC AGT (SEQ ID NO:19)
Primer 3 ACT GAG GAC TCA GGC ACC ACA GCC GGT GCT GCC CCA GGT TG
(SEQ?ID?NO:20)
Primer 4 TTT TTC TAG AGA AGC AGC AGC AGC CCA TG (SEQ ID NO:21)
Primer 1 and 2 is used to produce the sequence of volume from the extra p55 residue of 161-190.The foregoing basically condition of this PCR reaction is carried out, and uses the every kind of primer of 1 μ g and pUC-p55 as template.Equally, primer 3 and 3 is used to produce 3 '-end of TBP coding section and the connector between the coding hCG α subunit 5 ' end by PCR, use be that plasmid pSVLTBPhCG α is as template.Polyacrylamide gel electrophoresis on 8% gel (PAGE) has confirmed that the product of these PCR reactions for correct size (being respectively about 296bp and 121bp), uses the Wizard post to be purified then.Primer 2 and 3 design are such, and they contain overlap section, thus make these two kinds of PCR products (derive from primer 1 and 2 and derive from primer 3 and 4) can anneal is used to use the fusion PCR of primer 1 and 4.After the fusion reaction, will confirm and purifying with 1.5% sepharose and Wizard post to the expectation product of 400bp.Then this DNA is digested with Bgl II and Xba I, and be connected with the pSVLTBPhCG α of Bgl II/Xba I digestion.The existence of inset has obtained confirmation by the digestion with Bgl II and Xba I in isolating plasmid from the AG1 intestinal bacteria that transform.This new construct is named as pSVLTBP (20-190)-hCG α.
Equally, plasmid pSVLTBPhCG β is modified by Bgl II and the segmental replacement of Xcm I, still, this is that subclone by single PCR product carries out, rather than merges the PCR product.Used primer 1 and 2b (seeing below), and as the pUC-p55 of template.
Primer 2 b TTT TCC ACA GCC AGG GTG GCA TTG ATG GGG CGG CAC CGT GGA CCAGCA CCA GCT GTG GTG CCT GAG TCC TCA GTG (SEQ ID NO:22)
The PCR product (about 3376p) that produces is confirmed and purifying with aforesaid method,, connect to advance then among the pSVLTBPhCG β with Bgl II/Xba I digestion with Bgl II and the digestion of Xcm I.Existence to inset from the isolating plasmid of AG1 intestinal bacteria that derives from conversion confirms that by the digestion with Bgl II and Xcm I this novel constructs is named as pSVLTBP (20-190)-hCG β.
Subsequently this novel constructs is confirmed with dna sequencing.
Except producing these new is that these constructs are also advanced in other other expression vector that may be more suitable for stably express in CHO by subclone the plasmid of base with pSVL, and particularly carrier D2 was described to plasmid CLH3AXSV2DHFR (45) in the past.This is by converting side joint to Xho I site based on the BamH I site of the inset in the carrier of pSVL, excises this inset with the Xho I then and it is cloned among the D α of into Xhol digestion.
2, the moment of hybrid protein and stably express
Being used for moment expresses the COS-7 cell of TBP-hCG hybrid protein (ATCCCRL 1651, (47) that transfection ref.46) is to use electroporation to carry out.The COS-7 cell of exponential growth is removed by tryptic digestion, and by slightly centrifugal (800rpm, 4 minutes) collection, with cold phosphate buffered saline (PBS) (PBS), the pH7.3-7.4 washing is reunited again by centrifugal then.Is 5 * 10 with cell with the cold PBS of per 400 μ l 6The concentration of cell suspends again and mixes with 10 μ g plasmid DNA in the 2mm of precooling breach electroporation sulculus.For cotransfection, use every kind of plasmid of 35 μ g.Sulculus and cell at freezing again 10 minutes, are used BTX600 type instrument and 125V on ice then, and the condition of 950 μ F and R=8 is carried out electroporation.Afterwards cell is placed on ice and made it freezing in 10 minutes, be transferred to a 15ml and contain in the perfect medium of 9.5ml ambient temperature and (replenished Dulbecco ' s improvement Eagle ' the s substratum (DMEM) of the 1%L-glutamine of 10% calf serum (FBS)), and at room temperature placed 5 minutes.In this 15ml test tube after slight the mixing, be inoculated into whole inclusion on two P100 flat boards and place one 37 ℃, 5%CO 2In the incubator.After 18 hours, change substratum, and this new substratum only contains 1% or 0%FBS in some cases.After 72 hours, gather in the crops this conditioned medium, the centrifugal cell of removing is then-70 ℃ of refrigerated storages.
The transfection that is used for CHO-DUKX (CHO) cell of moment or stably express is to use the calcium phosphate precipitation method of DNA to carry out, preceding 24 hours of transfection, with the Chinese hamster ovary celI of exponential growth with every dull and stereotyped 7.5 * 10 5The density shop cloth of cell is on the 100mm culture plate.On the same day of transfection, 10 μ g plasmid DNA are put into 0.5ml transfection damping fluid (seeing below), add the 2M CaCl of 31 μ l 2, with this DNA-CaCl 2Solution carries out vortex mixed, and at room temperature places 45 minutes.After this, substratum is gone from the flat board suction, use an aseptic plastic pipe that DNA is added in the cell, and these cells were at room temperature placed 20 minutes, when finishing during this period, in flat board, add complete α (+) MEM that 5ml contains 10%FBS, with it at 37 ℃ of incubation 4-6 hours.Then substratum is inhaled from flat board and gone, these cells are carried out glycerine at 37 ℃ with the 15% glycerin liquid incubation 3.5 minutes in the transfection damping fluid impact.After this glycerine solution is removed, cell is washed twice with PBS, add the complete α of 10ml (+) MEM again, 10%FBS, and put back to 37 ℃ of incubators.For stable transfection, after 48 hours these cells were opened with selecting substratum (complete α (-) MEM (shortage nucleosides), the FBS and the 0.02 μ M methotrexate of 10% dialysis) to feed in 1: 10 minute.(non-resistance) cell of non-transfection is removed in week at 3-4 generally speaking, has been left transfection, the cell mass of methotrexate resistance.
3, the quantitative analysis of Biao Daing
The secretion of the hybrid protein of transfectional cell is to use the commercially available reagent box (R﹠amp that detects solubility p55; D Systems; Minneapolis Minnesota), detects according to the explanation of manufacturers.This detection also provides a kind of estimation to hybrid protein in conditioning and the substratum that handle, selects to be used for the dosage of biological detection based on this.
4, the assessment that heterodimer is formed
Be the ability of assessment TBP-hCG subunit syzygy combination and formation heterodimer, carried out using filled loaf immunodetection (Sandwichimmunoassay) at the antibody of hCG subunit.In this detection then, will be coated on the titer plate at the monoclonal antibody of hCG β subunit and be used for analyte and catch.Main detection antibody is a kind of goat polyclonal antibody, and it cultivates (#082422G-BiodesignInternational at people TSH α subunit; Kennenbunkport Maine), uses the anti-sheep polyclonal antibody of the rabbit (Cappel of horseradish peroxidase conversely; Durham, North Carolina) detects this goat antibody.
Use several different anti-hCG β subunit antibodies in this work, they are equipped with free α subunit and demonstrate detectable cross reactivity.One (3/6) in these antibody is used to commodity MAIAclonehCG detection kit (Biodata; Romt, Italy).
Height-protein bound droplet plate (Costar#3590) is coated with capture antibody, applies damping fluid (PBS, pH7.4,01mMCa with 100 μ l/ holes by incubation (2 hours, 37 ℃) ++, 0.1mM Mg ++) in 5 μ g/ml antibody-solutions apply.With washing soln (PBS, pH7.4+0.1%Tween20) washed once after, by with lock solution (3% bSA (BSA among the PBS; Fraction V-A-4503Sigma), pH7.4) hole is full of fully (about 400 μ l/ holes) and plate is sealed 37 ℃ of incubations 1 hour or at 4 ℃ of incubations that spend the night, then plate is used the lavation buffer solution washed twice, and with reference to and laboratory sample, in that (the 5mg/ml BSA among the PBS pH7.4) is diluted to behind the 100 μ l volumes and adds wherein with diluent.With plate and sample at 37 ℃ of incubations after 2 hours, with plate with washing soln washed twice again.Be added in dilute in the diluent 1: 5000 first detect antibody (100 μ l/ hole) and 37 ℃ of incubations 1 hour.Be added in the second detection antibody (the HRP link coupled is exempted from anti-sheep Ig) (100 μ l/ hole) that diluted in the diluent 1: 5000, and after 1 hour, plate washed three times with washing soln at 37 ℃ of incubations.Add 100 μ l tmb substrate solution (Kirkegaard and Perry laboratory), with plate incubation 20 minutes under the room temperature in the dark, by adding 50 μ l/ holes, 0.3M sulfuric acid stops enzyme reaction then.Use the droplet card reader plate to be analyzed then at wavelength 450nm place.
5, partial purification
In order better the activity of these hybrid egg matter to be carried out quantitatively with the immunosorption chromatography TBP-hCG hybrid protein being carried out partial purification.Used antibody be a kind of can be by commercial sources from R﹠amp; The monoclonal antibody that D System (MAB#225) obtains.Post is a CNBr activatory agarose, loads according to the explanation of manufacturers (Pharmacia).
Conditioned medium is to use 50 μ l SFM II substratum (GIBCO) of results every day from what collect the T-175 bottle converging of each clone, and each clone has 5 cuttings.Gleanings is carried out centrifugal (1000RPM) to remove cell debris.The test of commodity in use immunodetection is carried out the TBP content detection and is concentrated (Centricon un:ts by Arnicon this material then; Beverly Massachusetts), makes apparent TBP concentration be about 50ng/ml.
Make it have about 1MNaCl by adding NaCl and the electric conductivity of this solution being adjusted to the spissated TBP-hCG of 10ml (sample #188737) to 85mS/cm.With its anti--TBP immune affinity column by 0.5ml.Collect effluent and make it for the second time by this post.Wash this post with the 1M NaCl among the PBS afterwards.After with 50mM citric acid (pH2.5) wash-out, collect bonded TBP (20-161)-hCG.Elutriant (about 7ml) is concentrated by the filtration of using Amicon Centricon-10 ' s and carry out according to the explanation of manufacturers (Amicon), make it reach the volume of about 200 μ l.The PBS that adds about 800 μ l makes sample volume reach 1ml, and 4 ℃ of storages are until testing with bioassay method.
6, the active assessment of anti-TNF.
Multiple existing description of external TNF inductive cytotoxicity detection method that is used to assess the analogue of soluble TNF acceptor.We have used a kind of end user's breast cancer cell line.The detection method of BT-20 cell (ATCCHTB19).These cells are as existing describe (48) of the use on the basis of TNF biological detection.These cells have been replenished in RPMI 1640 substratum of 10% heat inactivation TBS 37 ℃ of cultivations, cell has been grown to maximum 80-90% converge, limited division in every like this 3-4 days, seed density is every T175cm 2Bottle about 3 * 10 6Individual cell.
BT-20 detects and to have used a kind of cell dye, Viola crystallina, painted, as handling the detection method that the back is assessed survivaling cell with TNF, dead cell can not absorb and keep this dyestuff with this.
In brief, it is as follows to be used to detect the active method of anti-TNF.Human TNF alpha (R﹠amp will recombinate; D systems) and laboratory sample be formulated in the substratum (RPMI 1640 and 5% heat-inactivated FBS, and be added in the hole on 96 well culture plates.Then with these cells with 1 * 10 5The density of cells/well tiles in these holes.The amount of the TNF α that adds is to measure in titration research previously, and represents the killed dosage of a kind of cell of about 50%.
After sample adds, cell was cultivated 48 hours at 39 ℃, afterwards, used violet staining and droplet card reader (570nm) to determine the ratio of viable cell.The result
1, the construct that is studied
To the summary of Design of the hybrid protein of research in down; To two kinds of control proteins, a kind of monomer solubility p55 (r-hTBP-1) and a kind of dimer TBP-immunoglobulin fusion proteins (TBP-IgG3) (basically as (10) as described in preparation) are studied, to compare.
The terminal counterpart that merges of the terminal TBPC-of construct TNPN-
R-hTBP-1 9 and 20 mixture 180 do not have
TBP-IgG3 9 and 20 mixture 190 IgG3 CH
TBP (20-161)-hCG 20 161 hCG α and hCG β (heterodimer)
(heterodimer) (heterodimer)
TBP (20-190)-hCG 20 190 hCG α and hCG β
The sequence of the DNA of coding TBP (20-190)-hCG and TBP (20-161)-hCG is shown in Fig. 1 and 2 respectively.Diagram summary to these constructs is shown in Fig. 3.
2, the proteinic secretion of TBP-hCG
Find that the transfected mammalian cell of all these constructs produces justacrine to substratum.The data of representing these results are shown in table 1 and table 2.
3, be assembled into TBP-hCG (α β) fused protein of heterodimer
The combination of TBP-hCG α and TBP-hCG β is to use the Layer cake detection method of hCG heterodimer to confirm, has only the co-transfection of α and β subunit fusions to produce heterodimer detection (table 3).
4, the TBP-hCG hybrid protein is compared with the TBP monomer and to be demonstrated the activity that has improved.
Kind of the protein that dyes that produces in COS-7 or Chinese hamster ovary celI is found to be in BT-20 gives birth to and detects and is the strong inhibition of TNF α.Some test result of samples are summarized in table 4.
Negative contrast (coming the conditioned medium of self simulation transfection) is included in the IX media samples.
Shown in Fig. 4-6 (point on the Y-axis), the adding of TNF (2.5ng/ml) causes the obvious reduction (being recorded by OD570) of viable count.Under each situation, the maximum protection usefulness that active sample had is that cell survival is returned to the level seen (, indicate the contrast of " having only cell ") when not adding TNF
Over against be protectiveness according to r-hTBP-1 and TBP-IgG3, demonstrate tangible dose-dependently, and the ED50 of r-hTBP-1 is about 100ng/ml (Fig. 4-6), the ED50 of TBP-TgG3 is about 1.5ng/ml (Fig. 4).
Demonstrate the protection of dose-dependently from the IX substratum or from the TBP-hCG construct (CHO or COS) of immune purifying thing, its ED50 is (Fig. 4-6) in the scope of about 2-11ng/ml.
External biological detects the results are described in table 5.These results show that this hybrid protein has suppressed the TNF cytotoxicity, and they are basically than TBP monomer effective force more.Activity is not protected in negative contrast.
Except TBP Dimerized strengthens the possibility of effectiveness, activity that also might this hybrid protein and the dimer of TBP interact, and it doesn't matter, but with sterically hindered relevant, this is because the partner interference soluble T BP/TNF of this hybrid protein and combining of cell surface TNF acceptor.
All reference that this paper quotes, comprise journal article or summary, disclosed or the corresponding U.S. or foreign patent application, the U.S. that has issued or foreign patent, or other any reference, the portion of closing is incorporated herein by reference, and comprises data whole in these reference, table, figure and text.In addition, the reference content of being quoted in this paper reference also all is incorporated herein.
To the reference of known method steps, conventional method steps, known method or conventional method and do not mean that we admit either side of the present invention, describe or embodiment is disclosed in these pertinent literatures, instruction or prompting.
Preamble has carried out complete disclosure to the description of specific embodiment to overall character of the present invention, other people use the knowledge (comprising the reference that this paper quotes) of art technology and can easily these specific embodiments be made amendment and adjust, and do not need to carry out too much experiment, but do not depart from overall content of the present invention.Thereby based on instruction and the indication of this paper, these adjustment and modification should be contained in embodiment disclosed herein and be equal within the meaning and the scope.The wording of this paper and term should be understood that in order more to know purpose of description, and not limit its scope.All speech of present disclosure and term should be made an explanation according to the instruction of this paper and indication and in conjunction with those of ordinary skills' existing knowledge by present technique field people's those of ordinary skill.
Table 1:COS-7 moment is expressed (TBP ELISA)
Hybrid protein Concentration (pg/ml)
TBP1 ????66
TBP-hCGα(20-161) ????5.1
TBP-hCGβ(20-161) ????0.5
TBP-hCG(20-161) ????2.7
Contrast ????＜0.25
Construct is to use pSVL (Pharmacia) to express
Table 2:COS-7 moment is expressed (TBP ELISA)
Hybrid protein Concentration (ng/ml)
TBP1 ????131
TBP-hCGα(20-190) ????81
TBP-hCGβ(20-190) ????9
TBP-hCG(20-190) ????62
Contrast ????＜1
Construct is to use carrier-pD alpha expression of containing the mouse metallothionein promoter
Table 3:COS-7 moment is expressed (detection of hCG dimer)
Hybrid protein Concentration (ng/ml)
?TBP1 ????＜0.2
?TBP-hCGα(20-190) ????＜0.2
?TBP-hCGβ(20-190) ????＜0.2
?TBP-hCG(20-190) ????38
Contrast ????＜0.2
Construct is to use the carrier-pD alpha expression of mouse metallothionein promoter
Table 4: to sample detection anti-TNF activity
Construct The cell source Properties of samples
r-hTBP-1 ????CHO Purifying
TBP-lgG3 ????CHO The IX conditioned medium
TBP(20-161)-hCG ????CHO The immunity purifying (anti--TBP)
TBP(20-190)-hCG ????CHO The IX conditioned medium
TBP(20-190)-hCG ????COS The IX conditioned medium
Table 5: to the entry evaluation of hybrid protein in the TNF cytotoxicity detects
Construct Merge partner Anti-TNF activity (ED50) * * in the BT-20 biological detection
r-hTBP-1 Do not have ????100ng/ml
TBP-lgG3 The lgG3 CH ????1.5ng/ml
TBP(20-161)-hCG HCG α and hCG β (heterodimer) ????2ng/ml
TBP(20-190)-hCG HCG α and hCG β (heterodimer) ????8-11ng/ml
The estimation quantitative and ED50 that * is used for the dosage of material is to use TBPELISA to carry out
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Sequence table (1) total information: (ⅰ) applicant:
(A) title: Applied Research Systems ARS Holding N. V
(B) street: 14 John B.Gorsiraweg
(C) city: Curacao
(D) country: Netherlands Antilles
(E) postcode:
(A) name: CAMPBELL, Robert C.
(B) street: 25 Meadowbrood Drive
(C) city: Wrentham
(D) state: Massachusetts
(E) country: USA
(A) name: JAMESON, Bradford A.
(B) street: 76 Robbins Street
(C) city: Milton
(D) state: Massachusetts
(E) country: USA
(A) name: CHAPPEL, Scott C.
(B) street: 125 Canton Avenue
(C) city: Milton
(D) state: Massachusetts
(E) country: USA (ⅱ) denomination of invention: hybrid protein (ⅲ) sequence number: 22 (ⅳ) mailing address:
(A) addressee: BROWDY AND NEIMARK
(B) street: 419 Seventh Street N.W., Ste.300
(C) city: Washington
(D) state: D.C.
(E) country: USA
(F) postcode: 22207 (ⅴ) computer-reader form:
(A) media type: floppy disk
(B) computer: IBM PC compatibility
(C) operating system: PC-DOS/MS-DOS
(D) software: PatentIn Release#1.0, Version#1.30 (ⅵ) is application materials formerly:
(A) application number: 60/011936
(B) applying date: on February 20th, 1996
(C) classification: (ⅷ) proxy's information:
(A) name: Browdy, Roger L.
(B) registration number: 25618
(C) case number: CAMPBELL=2A PCT (ⅸ) communications data:
(A) phone: (202) 628-5197
(B) fax: the information of (202) 737-3528 (2) SEQ ID NO:1: (ⅰ) sequence signature:
(A) length: 1049 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: cDNA (ⅸ) characteristics:
(A) title/keyword: CDS
(B) position: 278..1047 (ⅹ ⅰ) sequence description: SEQ ID NO:1TCCACATGGC TACAGGTAAG CGCCCCTAAA ATCCCTTTGG GCACAATGTG TCCTGAGGGG 60AGAGGCAGCG ACCTGTAGAT GGGACGGGGG CACTAACCCT CAGGTTTGGG GCTTCTCAAT 120CTCACTATCG CCATGTAAGC CCAGTATTTG GCCAATCTCA GAAAGCTCCT CCTCCCTGGA 180GGGATGGAGA GAGAAAAACA AACAGCTCCT GGAGCAGGGA GAGTGCTGGC CTCTTGCTCT 240CCGGCTCCCT CTGTTGCCCT CTGGTTTCTC CCCAGGC TCC CGG ACG TCC CTG CTC 295
Ser?Arg?Thr?Ser?Leu?Leu
1???????????????5CTG?GCT?TTT?GGC?CTG?CTC?TGC?CTG?CCC?TGG?CTT?CAA?GAG?GGC?AGT?GCC????343Leu?Ala?Phe?Gly?Leu?Leu?Cys?Leu?Pro?Trp?Leu?Gln?Glu?Gly?Ser?Ala
10??????????????????15??????????????????20GAT?AGT?GTG?TGT?CCC?CAA?GGA?AAA?TAT?ATC?CAC?CCT?CAA?AAT?AAT?TCC????391Asp?Ser?Val?Cys?Pro?Gln?Gly?Lys?Tyr?Ile?His?Pro?Gln?Asn?Asn?Ser
25??????????????????30??????????????????35ATT?TGC?TGT?ACC?AAG?TGC?CAC?AAA?GGA?ACC?TAC?TTG?TAC?AAT?GAC?TGT????439Ile?Cys?Cys?Thr?Lys?Cys?His?Lys?Gly?Thr?Tyr?Leu?Tyr?Asn?Asp?Cys
40??????????????????45??????????????????50CCA?GGC?CCG?GGG?CAG?GAT?ACG?GAC?TGC?AGG?GAG?TGT?GAG?AGC?GGC?TCC????487Pro?Gly?Pro?Gly?Gln?Asp?Thr?Asp?Cys?Arg?Glu?Cys?Glu?Ser?Gly?Ser?55??????????????????60??????????????????65??????????????????70TTC?ACC?GCT?TCA?GAA?AAC?CAC?CTC?AGA?CAC?TGC?CTC?AGC?TGC?TCC?AAA????535Phe?Thr?Ala?Ser?Glu?Asn?His?Leu?Arg?His?Cys?Leu?Ser?Cys?Ser?Lys
75??????????????????80??????????????????85TGC?CGA?AAG?GAA?ATG?GGT?CAG?GTG?GAG?ATC?TCT?TCT?TGC?ACA?GTG?GAC????583Cys?Arg?Lys?Glu?Met?Gly?Gln?Val?Glu?Ile?Ser?Ser?Cys?Thr?Val?Asp
90??????????????????95?????????????????100CGG?GAC?ACC?GTG?TGT?GGC?TGC?AGG?AAG?AAC?CAG?TAC?CGG?CAT?TAT?TGG????631Arg?Asp?Thr?Val?Cys?Gly?Cys?Arg?Lys?Asn?Gln?Tyr?Arg?His?Tyr?Trp
105?????????????????110?????????????????115AGT?GAA?AAC?CTT?TTC?CAG?TGC?TTC?AAT?TGC?AGC?CTC?TGC?CTC?AAT?GGG????679Ser?Glu?Asn?Leu?Phe?Gln?Cys?Phe?Asn?Cys?Ser?Leu?Cys?Leu?Asn?Gly
120?????????????????125?????????????????130ACC?GTG?CAC?CTC?TCC?TGC?CAG?GAG?AAA?CAG?AAC?ACC?GTG?TGC?ACC?TGC????727Thr?Val?His?Leu?Ser?Cys?Gln?Glu?Lys?Gln?Asn?Thr?Val?Cys?Thr?Cys135?????????????????140?????????????????145?????????????????150CAT?GCA?GGT?TTC?TTT?CTA?AGA?GAA?AAC?GAG?TGT?GTC?TCC?TGT?GCC?GGT????775His?Ala?Gly?Phe?Phe?Leu?Arg?Glu?Asn?Glu?Cys?Val?Ser?Cys?Ala?Gly
155?????????????????????160?????????????????165GCT?GCC?CCA?GGT?TGC?CCA?GAA?TGC?ACG?CTA?CAG?GAA?AAC?CCA?TTC?TTC????823Ala?Ala?Pro?Gly?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro?Phe?Phe
170?????????????????175?????????????????180TCC?CAG?CCG?GGT?GCC?CCA?ATA?CTT?CAG?TGC?ATG?GGC?TGC?TGC?TTC?TCT???871Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys?Phe?Ser
185?????????????????190?????????????????195AGA?GCA?TAT?CCC?ACT?CCA?CTA?AGG?TCC?AAG?AAG?ACG?ATG?TTG?GTC?CAA???919Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu?Val?Gln
200?????????????????205?????????????????210AAG?AAC?GTC?ACC?TCA?GAG?TCC?ACT?TGC?TGT?GTA?GCT?AAA?TCA?TAT?AAC???967Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser?Tyr?Asn215?????????????????220?????????????????225?????????????????230AGG?GTC?ACA?GTC?ATG?GGG?GGT?TTC?AAA?GTG?GAG?AAC?CAC?ACG?GGG?TGC??1015Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr?Gly?Cys
235?????????????????240?????????????????245CAC?TGC?AGT?ACT?TGT?TAT?TAT?CAC?AAA?TCT??TA?AG???????????????????1049His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
The information of 250 255 (2) SEQ ID NO:2: (ⅰ) sequence signature:
(A) length: 256 amino acid
(B) type: amino acid
(C) topological framework: linear (ⅱ) molecule type: protein (ⅹ ⅰ) sequence description: SEQ ID NO:2Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu Cys Leu Pro Trp 15 10 15Leu Gln Glu Gly Ser Ala Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile
20??????????????????25??????????????????30His?Pro?Gln?Asn?Asn?Ser?Ile?Cys?Cys?Thr?Lys?Cys?His?Lys?Gly?Thr
35??????????????????40??????????????????45Tyr?Leu?Tyr?Asn?Asp?Cys?Pro?Gly?Pro?Gly?Gln?Asp?Thr?Asp?Cys?Arg
50??????????????????55??????????????????60Glu?Cys?Glu?Ser?Gly?Ser?Phe?Thr?Ala?Ser?Glu?Asn?His?Leu?Arg?His?65??????????????????70??????????????????75??????????????????80Cys?Leu?Ser?Cys?Ser?Lys?Cys?Arg?Lys?Glu?Met?Gly?Gln?Val?Glu?Ile
85??????????????????90??????????????????95Ser?Ser?Cys?Thr?Val?Asp?Arg?Asp?Thr?Val?Cys?Gly?Cys?Arg?Lys?Asn
100?????????????????105?????????????????110Gln?Tyr?Arg?His?Tyr?Trp?Ser?Glu?Asn?Leu?Phe?Gln?Cys?Phe?Asn?Cys
115?????????????????120?????????????????125Ser?Leu?Cys?Leu?Asn?Gly?Thr?Val?His?Leu?Ser?Cys?Gln?Glu?Lys?Gln
130?????????????????135?????????????????140Asn?Thr?Val?Cys?Thr?Cys?His?Ala?Gly?Phe?Phe?Leu?Arg?Glu?Asn?Glu145?????????????????150?????????????????155?????????????????160Cys?Val?Ser?Cys?Ala?Gly?Ala?Ala?Pro?Gly?Cys?Pro?Glu?Cys?Thr?Leu
165?????????????????170?????????????????175Gln?Glu?Asn?Pro?Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys
180?????????????????185?????????????????190Met?Gly?Cys?Cys?Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys
195?????????????????200?????????????????205Lys?Thr?Met?Leu?Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys
210?????????????????215?????????????????220Val?Ala?Lys?Ser?Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val225?????????????????230?????????????????235?????????????????240Glu?Asn?His?Thr?Gly?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
The information of 245 250 255 (2) SEQ ID NO:3: (ⅰ) sequence signature:
(A) length: 1202 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: cDNA (ⅸ) characteristics:
(A) title/keyword: CDS
(B) position: 279..1199 (ⅹ ⅰ) sequence description: SEQ ID NO:3CTCGAGATGG CTACAGGTAA GCGCCCCTAA AATCCCTTTG GGCACAATGT GTCCTGAGGG 60GAGAGGTAGC GACCTGTAGA TGGGACGGGG GCACTAACCC TGAGGTTTGG GGCTTCTGAA 120TGTGAGTATC GCCATGTAAG CCCAGTATTT GGCCAATGTC AGAAAGCTCC TGGTCCCTGG 180AGGGATGGAG AGAGAAAAAC AAACAGCTCC TGGAGCAGGG AGAGTGCTGG CCTCTTGCTC 240TCCGGCTCCC TCTGTTGCCC TGTGGTTTCT CCCCAGGC TCC CGG ACG TCC CTG 293
Ser?Arg?Thr?Ser?Leu
260CTC?CTG?GCT?TTT?GGC?CTG?CTC?TGC?CTG?CCC?TGG?CTT?CAA?GAG?GGC?AGT????341Leu?Leu?Ala?Phe?Gly?Leu?Leu?Cys?Leu?Pro?Trp?Leu?Gln?Glu?Gly?Ser
265?????????????????270?????????????????275GCC?GAT?AGT?GTG?TGT?CCC?CAA?GGA?AAA?TAT?ATC?CAC?CCT?CAA?AAT?AAT????389Ala?Asp?Ser?Val?Cys?Pro?Gln?Gly?Lys?Tyr?Ile?His?Pro?Gln?Asn?Asn
280?????????????????285?????????????????290TCG?ATT?TGC?TGT?ACC?AAG?TGC?CAC?AAA?GGA?ACC?TAC?TTG?TAC?AAT?GAC????437Ser?Ile?Cys?Cys?Thr?Lys?Cys?His?Lys?Gly?Thr?Tyr?Leu?Tyr?Asn?Asp
295?????????????????300??????????????????305TGT?CCA?GGC?CCG?GGG?CAG?GAT?ACG?GAC?TGC?AGG?GAG?TGT?GAG?AGC?GGC????485Cys?Pro?Gly?Pro?Gly?Gln?Asp?Thr?Asp?Cys?Arg?Glu?Cys?Glu?Ser?Gly310?????????????????315?????????????????320?????????????????325TCT?TTC?ACC?GCT?TCA?GAA?AAC?CAC?CTC?AGA?CAC?TGC?CTC?AGC?TGC?TCC????533Ser?Phe?Thr?Ala?Ser?Glu?Asn?His?Leu?Arg?His?Cys?Leu?Ser?Cys?Ser
330?????????????????335?????????????????340AAA?TGC?CGA?AAG?GAA?ATG?GGT?CAG?GTG?GAG?ATC?TCT?TCT?TGC?ACA?GTG????581Lys?Cys?Arg?Lys?Glu?Met?Gly?Gln?Val?Glu?Ile?Ser?Ser?Cys?Thr?Val
345?????????????????350?????????????????355GAC?CGG?GAC?ACC?GTG?TGT?GGC?TGC?AGG?AAG?AAC?CAG?TAC?CGG?CAT?TAT???629Asp?Arg?Asp?Thr?Val?Cys?Gly?Cys?Arg?Lys?Asn?Gln?Tyr?Arg?His?Tyr
360?????????????????365?????????????????370TGG?AGT?GAA?AAC?CTT?TTC?CAG?TGC?TTC?AAT?TGC?AGC?CTC?TGC?CTC?AAT???677Trp?Ser?Glu?Asn?Leu?Phe?Gln?Cys?Phe?Asn?Cys?Ser?Leu?Cys?Leu?Asn
375?????????????????380?????????????????385GGG?ACC?GTG?CAC?CTC?TCC?TGC?CAG?GAG?AAA?CAG?AAC?ACC?GTG?TGC?ACC???725Gly?Thr?Val?His?Leu?Ser?Cys?Gln?Glu?Lys?Gln?Asn?Thr?Val?Cys?Thr390?????????????????395?????????????????400?????????????????405TGC?CAT?GCA?GGT?TTC?TTT?CTA?AGA?GAA?AAC?GAG?TGT?GTC?TCC?TGT?GCT???773Cys?His?Ala?Gly?Phe?Phe?Leu?Arg?Glu?Asn?Glu?Cys?Val?Ser?Cys?Ala
410?????????????????415?????????????????420GGT?GCT?GGT?CCA?CGG?TGC?CGC?CCC?ATC?AAT?GCC?ACC?CTG?GCT?GTG?GAG???821Gly?Ala?Gly?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu?Ala?Val?Glu
425?????????????????430?????????????????435AAG?GAG?GGC?TGC?CCC?GTG?TGC?ATC?ACC?GTC?AAC?ACC?ACC?ATC?TGT?GCC???869Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr?Ile?Cys?Ala
440?????????????????445?????????????????450GGC?TAC?TGC?CCC?ACC?ATG?ACC?CGC?GTG?CTG?CAG?GGG?GTC?CTC?CCC?GCC???917Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val?Leu?Pro?Ala
455?????????????????460?????????????????465CTG?CCT?CAG?GTG?GTG?TGC?AAC?TAC?CGC?GAT?GTG?CGC?TTC?GAG?TCC?ATC???965Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe?Glu?Ser?Ile470?????????????????475?????????????????480?????????????????485CGG?CTC?CCT?GGC?TGC?CCG?CGC?GGC?GTG?AAC?CCC?GTG?GTC?TCC?TAC?GCT??1013Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Asn?Pro?Val?Val?Ser?Tyr?Ala
490?????????????????495?????????????????500GTG?GCT?CTC?AGC?TGT?CAA?TGT?GCA?CTC?TGC?CGC?CGC?AGC?ACC?ACT?GAC??1061Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Ser?Thr?Thr?Asp
505??????????????????510?????????????????515TGC?GGG?GGT?CCC?AAG?GAC?CAC?CCC?TTG?ACC?TGT?GAT?GAC?CCC?CGC?TTC??1109Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp?Pro?Arg?Phe
520?????????????????525?????????????????530CAG?GAC?TCC?TCT?TCC?TCA?AAG?GCC?CCT?CCC?CCC?AGC?CTT?CCA?AGC?CCA??1157Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu?Pro?Ser?Pro
535?????????????????540?????????????????545TCC?CGA?CTC?CCG?GGG?CCC?TCG?GAC?ACC?CCG?ATC?CTC?CCA?CAA?TAA??????1202Ser?Arg?Leu?Pro?Gly?Pro?Ser?Asp?Thr?Pro?Ile?Leu?Pro?Gln550?????????????????555?????????????????560
(2) information of SEQ ID NO:4:
(ⅰ) sequence signature:
(A) length: 307 amino acid
(B) type: amino acid
(C) topological framework: linearity
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:4Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu Cys Leu Pro Trp 15 10 15Leu Gln Glu Gly Ser Ala Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile
20??????????????????25??????????????????30His?Pro?Gln?Asn?Asn?Ser?Ile?Cys?Cys?Thr?Lys?Cys?His?Lys?Gly?Thr
35??????????????????40??????????????????45Tyr?Leu?Tyr?Asn?Asp?Cys?Pro?Gly?Pro?Gly?Gln?Asp?Thr?Asp?Cys?Arg
50??????????????????55??????????????????60Glu?Cys?Glu?Ser?Gly?Ser?Phe?Thr?Ala?Ser?Glu?Asn?His?Leu?Arg?His?65??????????????????70??????????????????75??????????????????80Cys?Leu?Ser?Cys?Ser?Lys?Cys?Arg?Lys?Glu?Met?Gly?Gln?Val?Glu?Ile
85??????????????????90??????????????????95Ser?Ser?Cys?Thr?Val?Asp?Arg?Asp?Thr?Val?Cys?Gly?Cys?Arg?Lys?Asn
100?????????????????105?????????????????110Gln?Tyr?Arg?His?Tyr?Trp?Ser?Glu?Asn?Leu?Phe?Gln?Cys?Phe?Asn?Cys
115?????????????????120?????????????????125Ser?Leu?Cys?Leu?Asn?Gly?Thr?Val?His?Leu?Ser?Cys?Gln?Glu?Lys?Gln
130?????????????????135?????????????????140Asn?Thr?Val?Cys?Thr?Cys?His?Ala?Gly?Phe?Phe?Leu?Arg?Glu?Asn?Glu145?????????????????150?????????????????155?????????????????160Cys?Val?Ser?Cys?Ala?Gly?Ala?Gly?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala
165?????????????????170?????????????????175Thr?Leu?Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn
180?????????????????185?????????????????190Thr?Thr?Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln
195?????????????????200?????????????????205Gly?Val?Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val
210?????????????????215?????????????????220Arg?Phe?Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Asn?Pro225?????????????????230?????????????????235?????????????????240Val?Val?Ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg
245?????????????????250?????????????????255Arg?Ser?Thr?Thr?Asp?Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys
260?????????????????265?????????????????270Asp?Asp?Pro?Arg?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro
275?????????????????280?????????????????285Ser?Leu?Pro?Ser?Pro?Ser?Arg?Leu?Pro?Gly?Pro?Ser?Asp?Thr?Pro?Ile
290?????????????????295?????????????????300Leu?Pro?Gln305
(2) information of SEQ ID NO:5:
(ⅰ) sequence signature:
(A) length: 1147 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
(ⅸ) characteristics:
(A) title/keyword: CDS
(B) position: 278..1132
(ⅹ ⅰ) sequence description: SEQ ID NO:5TCGAGATGGC TACAGGTAAG CGCCCCTAAA ATCCCTTTGG GCACAATGTG TCCTGAGGGG 60AGAGGCAGCG ACCTGTAGAT GGGACGGGGG CACTAACCCT CAGGTTTGGG GCTTTTGAAT 120GTGAGTATGG CCATGTAAGC CCAGTATTTG CCCAATCTCA GAAAGCTCCT GGTCCCTGGA 180GGGATGGAGA GAGAAAAACA AACAGCTCCT GGAGCAGGGA CACTCCTGGC CTCTTGCTCT 240GCGGCTCCGT GTGTTGCCCT GTGGTTTCTC CCCACGC TCC CGG ACG TCC CTG CTC 295
Ser?Arg?Thr?Ser?Leu?Leu
310CTG?GCT?TTT?GGC?CTG?CTC?TGC?CTG?CCC?TGG?CTT?CAA?GAG?GGC?AGT?GCC????343Leu?Ala?Phe?Gly?Leu?Leu?Cys?Leu?Pro?Trp?Leu?Gln?Glu?Gly?Ser?Ala
315?????????????????320?????????????????325GAT?AGT?GTG?TGT?CCC?CAA?GGA?AAA?TAT?ATC?CAC?CCT?CAA?AAT?AAT?TCG????391Asp?Ser?Val?Cys?Pro?Gln?Gly?Lys?Tyr?Ile?His?Pro?Gln?Asn?Asn?Ser330?????????????????335?????????????????340?????????????????345ATT?TGC?TGT?ACC?AAG?TGC?CAC?AAA?GGA?ACC?TAC?TTG?TAC?AAT?GAC?TGT????439Ile?Cys?Cys?Thr?Lys?Cys?His?Lys?Gly?Thr?Tyr?Leu?Tyr?Asn?Asp?Cys
350?????????????????355?????????????????360CCA?GGC?CCG?GGG?CAG?GAT?ACC?GAC?TGC?AGG?GAG?TGT?GAG?AGC?GGC?TCC????487Pro?Gly?Pro?Gly?Gln?Asp?Thr?Asp?Cys?Arg?Glu?Cys?Glu?Ser?Gly?Ser
365?????????????????370?????????????????375TTC?ACC?GCT?TCA?GAA?AAC?CAC?CTC?AGA?CAC?TGC?CTC?AGC?TGC?TCC?AAA????535Phe?Thr?Ala?Ser?Glu?Asn?His?Leu?Arg?His?Cys?Leu?Ser?Cys?Ser?Lys
380?????????????????385?????????????????390TGC?CGA?AAG?GAA?ATG?GGT?CAG?GTG?GAG?ATC?TCT?TCT?TGC?ACA?GTG?GAC????583Cys?Arg?Lys?Glu?Met?Gly?Gln?Val?Glu?Ile?Ser?Ser?Cys?Thr?Val?Asp
395?????????????????400?????????????????405CGG?GAC?ACC?GTG?TGT?GGC?TGC?AGG?AAG?AAC?CAG?TAC?CGG?CAT?TAT?TGG????631Arg?Asp?Thr?Val?Cys?Gly?Cys?Arg?Lys?Asn?Gln?Tyr?Arg?His?Tyr?Trp410?????????????????415?????????????????420?????????????????425AGT?GAA?AAC?CTT?TTC?CAG?TGC?TTC?AAT?TGC?ACC?CTC?TGC?CTC?AAT?GGG????679Ser?Glu?Asn?Leu?Phe?Gln?Cys?Phe?Asn?Cys?Thr?Leu?Cys?Leu?Asn?Gly
430?????????????????435?????????????????440ACC?GTG?CAC?CTC?TCC?TGT?CAG?GAG?AAA?CAG?AAC?ACC?GTC?TGC?ACC?TGC????727Thr?Val?His?Leu?Ser?Cys?Gln?Glu?Lys?Gln?Asn?Thr?Val?Cys?Thr?Cys
445?????????????????450?????????????????455CAT?GCA?GGT?TTC?TTT?CTA?AGA?GAA?AAC?GAG?TGT?GTC?TCC?TGT?AGT?AAC????775His?Ala?Gly?Phe?Phe?Leu?Arg?Glu?Asn?Glu?Cys?Val?Ser?Cys?Ser?Asn
460?????????????????465?????????????????470TGT?AAG?AAA?AGC?CTG?GAG?TGC?ACG?AAG?TTG?TCC?CTA?CCC?CAG?ATT?GAG????823Cys?Lys?Lys?Ser?Leu?Glu?Cys?Thr?Lys?Leu?Ser?Leu?Pro?Gln?Ile?Glu
475?????????????????480?????????????????485AAT?GTT?AAG?GGC?ACT?GAG?GAC?TCA?GGC?ACC?ACA?GCC?GGT?GCT?GCC?CCA????871Asn?Val?Lys?Gly?Thr?Glu?Asp?Ser?Gly?Thr?Thr?Ala?Gly?Ala?Ala?Pro490?????????????????495?????????????????500?????????????????505GGT?TGC?CCA?GAA?TGC?ACG?CTA?CAG?GAA?AAC?CCA?TTC?TTC?TCC?CAG?CCG???919Gly?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro?Phe?Phe?Ser?Gln?Pro
510?????????????????515?????????????????520GGT?GCC?CCA?ATA?CTT?CAG?TGC?ATG?GGC?TGC?TGC?TTC?TCT?AGA?GCA?TAT???967Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys?Phe?Ser?Arg?Ala?Tyr
525?????????????????530?????????????????535CCC?ACT?CCA?CTA?AGG?TCC?AAG?AAG?ACG?ATG?TTG?GTC?CAA?AAG?AAC?GTC??1015Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu?Val?Gln?Lys?Asn?Val
540?????????????????545?????????????????550ACC?TCA?GAG?TCC?ACT?TGC?TGT?GTA?GCT?AAA?TCA?TAT?AAC?AGG?GTC?ACA??1063Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser?Tyr?Asn?Arg?Val?Thr
555?????????????????560?????????????????565GTA?ATG?GGG?GGT?TTC?AAA?GTG?GAG?AAC?CAC?ACG?GCG?TGC?CAC?TGC?AGT??1111Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr?Ala?Cys?His?Cys?Ser570?????????????????575?????????????????580?????????????????585ACT?TGT?TAT?TAT?CAC?AAA?TCT?TAAGGATCCC?TCGAG?????????????????????1147Thr?Cys?Tyr?Tyr?His?Lys?Ser
590
(2) information of SEQ ID NO:6:
(ⅰ) sequence signature:
(A) length: 285 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:6Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu Cys Leu Pro Trp 15 10 15Leu Gln Glu Gly Ser Ala Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile
20??????????????????25??????????????????30His?Pro?Gln?Asn?Asn?Ser?Ile?Cys?Cys?Thr?Lys?Cys?His?Lys?Gly?Thr
35??????????????????40??????????????????45Tyr?Leu?Tyr?Asn?Asp?Cys?Pro?Gly?Pro?Gly?Gln?Asp?Thr?Asp?Cys?Arg
50??????????????????55??????????????????60Glu?Cys?Glu?Ser?Gly?Ser?Phe?Thr?Ala?Ser?Glu?Asn?His?Leu?Arg?His?65??????????????????70??????????????????75??????????????????80Cys?Leu?Ser?Cys?Ser?Lys?Cys?Arg?Lys?Glu?Met?Gly?Gln?Val?Glu?Ile
85??????????????????90??????????????????95Ser?Ser?Cys?Thr?Val?Asp?Arg?Asp?Thr?Val?Cys?Gly?Cys?Arg?Lys?Asn
100?????????????????105?????????????????110Gln?Tyr?Arg?His?Tyr?Trp?Ser?Glu?Asn?Leu?Phe?Gln?Cys?Phe?Asn?Cys
115?????????????????120?????????????????125Thr?Leu?Cys?Leu?Asn?Gly?Thr?Val?His?Leu?Ser?Cys?Gln?Glu?Lys?Gln
130?????????????????135?????????????????140Asn?Thr?Val?Cys?Thr?Cys?His?Ala?Gly?Phe?Phe?Leu?Arg?Glu?Asn?Glu145?????????????????150?????????????????155?????????????????160Cys?Val?Ser?Cys?Ser?Asn?Cys?Lys?Lys?Ser?Leu?Glu?Cys?Thr?Lys?Leu
165?????????????????170?????????????????175Ser?Leu?Pro?Gln?Ile?Glu?Asn?Val?Lys?Gly?Thr?Glu?Asp?Ser?Gly?Thr
180?????????????????185?????????????????190Thr?Ala?Gly?Ala?Ala?Pro?Gly?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn
195?????????????????200?????????????????205Pro?Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys
210?????????????????215?????????????????220Cys?Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met225?????????????????230?????????????????235?????????????????240Leu?Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys
245?????????????????250?????????????????255Ser?Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His
260?????????????????265?????????????????270Thr?Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
275?????????????????280?????????????????285
(2) information of SEQ ID NO:7:
(ⅰ) sequence signature:
(A) length: 1301 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
(ⅸ) characteristics:
(A) title/keyword: CDS
(B) position: 279..1287
(ⅹ ⅰ) sequence description: SEQ ID NO:7CTCGAGATGG CTACAGGTAA GCGCCCCTAA AATCCCTTTG GGCACAATGT GTCCTGAGGG 60GAGAGGCAGC GACCTGTAGA TGGGACGGGG GCACTAACCC TCAGGTTTGG GGCTTCTGAA 120TGTGAGTATC GCCATGTAAG CCCAGTATTT GGCCAATGTC AGAAAGCTCC TGGTCCCTGG 180AGGGATGGAG AGAGAAAAAC AAACACCTCC TGGAGCAGGG AGAGTGCTGC CCTCTTGCTC 240TCCGGCTCCC TCTGTTGCCC TCTGGTTTCT CCCCAGGC TCC CGG ACG TCC CTG 293
Ser?Arg?Thr?Ser?Leu
290CTC?CTG?GCT?TTT?GGC?CTG?CTC?TGC?CTG?CCC?TGG?CTT?CAA?GAG?GGC?AGT????341Leu?Leu?Ala?Phe?Gly?Leu?Leu?Cys?Leu?Pro?Trp?Leu?Gln?Glu?Gly?Ser
295?????????????????300?????????????????305GCC?GAT?AGT?GTG?TGT?CCC?CAA?GGA?AAA?TAT?ATC?CAC?CCT?CAA?AAT?AAT????389Ala?Asp?Ser?Val?Cys?Pro?Gln?Gly?Lys?Tyr?Ile?His?Pro?Gln?Asn?Asn
310?????????????????315?????????????????320TCG?ATT?TGC?TGT?ACC?AAG?TGC?CAC?AAA?GGA?ACC?TAC?TTG?TAC?AAT?GAC????437Ser?Ile?Cys?Cys?Thr?Lys?Cys?His?Lys?Gly?Thr?Tyr?Leu?Tyr?Asn?Asp
325?????????????????330?????????????????335TGT?CCA?GGC?CCG?GGG?CAG?GAT?ACG?GAC?TGC?AGG?GAG?TGT?GAG?AGC?GGC????485Cys?Pro?Gly?Pro?Gly?Gln?Asp?Thr?Asp?Cys?Arg?Glu?Cys?Glu?Ser?Gly
340?????????????????345?????????????????350TCC?TTC?ACC?GCT?TCA?GAA?AAC?CAC?CTC?AGA?CAC?TGC?CTC?AGC?TGC?TCC???533Ser?Phe?Thr?Ala?Ser?Glu?Asn?His?Leu?Arg?His?Cys?Leu?Ser?Cys?Ser355?????????????????360?????????????????365?????????????????370AAA?TGC?CGA?AAG?GAA?ATG?GGT?CAG?GTG?GAG?ATC?TCT?TCT?TGC?ACA?GTG???581Lys?Cys?Arg?Lys?Glu?Met?Gly?Gln?Val?Glu?Ile?Ser?Ser?Cys?Thr?Val
375?????????????????380?????????????????385GAC?CGG?GAC?ACC?GTG?TGT?GGC?TGC?AGG?AAG?AAC?CAG?TAC?CGG?CAT?TAT???629Asp?Arg?Asp?Thr?Val?Cys?Gly?Cys?Arg?Lys?Asn?Gln?Tyr?Arg?His?Tyr
390?????????????????395?????????????????400TGG?AGT?GAA?AAC?CTT?TTC?CAG?TGC?TTC?AAT?TGC?AGC?CTC?TGC?CTC?AAT???677Trp?Ser?Glu?Asn?Leu?Phe?Gln?Cys?Phe?Asn?Cys?Ser?Leu?Cys?Leu?Asn
405?????????????????410?????????????????415GGG?ACC?GTG?CAC?CTC?TCC?TGC?CAG?GAG?AAA?CAG?AAC?ACC?GTG?TGC?ACC???725Gly?Thr?Val?His?Leu?Ser?Cys?Gln?Glu?Lys?Gln?Asn?Thr?Val?Cys?Thr
420?????????????????425?????????????????430TGC?CAT?GCA?GGT?TTC?TTT?CTA?AGA?GAA?AAC?GAG?TGT?GTC?TCC?TGT?AGT???773Cys?His?Ala?Gly?Phe?Phe?Leu?Arg?Glu?Asn?Glu?Cys?Val?Ser?Cys?Ser435?????????????????440?????????????????445?????????????????450AAC?TGT?AAG?AAA?AGC?CTG?GAG?TGC?ACG?AAG?TTG?TGC?CTA?CCC?CAG?ATT???821Asn?Cys?Lys?Lys?Ser?Leu?Glu?Cys?Thr?Lys?Leu?Cys?Leu?Pro?Gln?Ile
455?????????????????460?????????????????465GAG?AAT?GTT?AAG?GGC?ACT?GAG?GAC?TCA?GGC?ACC?ACA?GCT?GGT?GCT?GGT???869Glu?Asn?Val?Lys?Gly?Thr?Glu?Asp?Ser?Gly?Thr?Thr?Ala?Gly?Ala?Gly
470?????????????????475?????????????????480CCA?CGG?TGC?CGC?CCC?ATC?AAT?GCC?ACC?CTG?GCT?GTG?GAG?AAG?GAG?GGC???917Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu?Ala?Val?Glu?Lys?Glu?Gly
485?????????????????490?????????????????495TGC?CCC?GTG?TGC?ATC?ACC?GTC?AAC?ACC?ACC?ATC?TGT?GCC?GGC?TAC?TGC???965Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr?Ile?Cys?Ala?Gly?Tyr?Cys
500?????????????????505?????????????????510CCC?ACC?ATG?ACC?CGC?GTG?CTG?CAG?GGG?GTC?CTG?CCG?GCC?CTG?CCT?CAG??1013Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val?Leu?Pro?Ala?Leu?Pro?Gln515?????????????????520?????????????????525?????????????????530GTG?GTG?TGC?AAC?TAC?CGC?GAT?GTG?CGC?TTC?GAG?TCC?ATC?CGG?CTC?CCT??1061Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe?Glu?Ser?Ile?Arg?Leu?Pro
535?????????????????540?????????????????545GGC?TGC?CCG?CGC?GGC?GTG?AAC?CCC?GTG?GTC?TCC?TAC?GCC?GTG?GCT?CTC??1109Gly?Cys?Pro?Arg?Gly?Val?Asn?Pro?Val?Val?Ser?Tyr?Ala?Val?Ala?Leu
550?????????????????555?????????????????560AGC?TGT?CAA?TGT?GCA?CTC?TGC?CGC?CGC?AGC?ACC?ACT?GAC?TGC?GGG?GGT??1157Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Ser?Thr?Thr?Asp?Cys?Gly?Gly
565?????????????????570?????????????????575CCC?AAG?GAC?CAC?CCC?TTG?ACC?TGT?GAT?GAC?CCC?CGC?TTC?CAG?GAC?TCC??1205Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp?Pro?Arg?Phe?Gln?Asp?Ser
580?????????????????585?????????????????590TCT?TCC?TCA?AAG?GCC?CCT?CCC?CCC?AGC?CTT?CCA?AGC?CCA?TCC?CGA?CTC??1253Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu?Pro?Ser?Pro?Ser?Arg?Leu595?????????????????600?????????????????605?????????????????610CCG?GGG?CCC?TCG?GAC?ACC?CCG?ATC?CTC?CCA?CAA?T?AAGGATCCCT?CGAG????1301Pro?Gly?Pro?Ser?Asp?Thr?Pro?Ile?Leu?Pro?Gln
615?????????????????620
(2) information of SEQ ID NO:8:
(ⅰ) sequence signature:
(A) length: 336 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: SEQ ID NO:8Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu Cys Leu Pro Trp 15 10 15Leu Gln Glu Gly Ser Ala Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile
20??????????????????25??????????????????30His?Pro?Gln?Asn?Asn?Ser?Ile?Cys?Cys?Thr?Lys?Cys?His?Lys?Gly?Thr
35??????????????????40??????????????????45Tyr?Leu?Tyr?Asn?Asp?Cys?Pro?Gly?Pro?Gly?Gln?Asp?Thr?Asp?Cys?Arg
50??????????????????55??????????????????60Glu?Cys?Glu?Ser?Gly?Ser?Phe?Thr?Ala?Ser?Glu?Asn?His?Leu?Arg?His?65??????????????????70??????????????????75??????????????????80Cys?Leu?Ser?Cys?Ser?Lys?Cys?Arg?Lys?Glu?Met?Gly?Gln?Val?Glu?Ile
85??????????????????90??????????????????95Ser?Ser?Cys?Thr?Val?Asp?Arg?Asp?Thr?Val?Cys?Gly?Cys?Arg?Lys?Asn
100?????????????????105?????????????????110Gln?Tyr?Arg?His?Tyr?Trp?Ser?Glu?Asn?Leu?Phe?Gln?Cys?Phe?Asn?Cys
115?????????????????120?????????????????125Ser?Leu?Cys?Leu?Asn?Gly?Thr?Val?His?Leu?Ser?Cys?Gln?Glu?Lys?Gln
130?????????????????135?????????????????140Asn?Thr?Val?Cys?Thr?Cys?His?Ala?Gly?Phe?Phe?Leu?Arg?Glu?Asn?Glu145?????????????????150?????????????????155?????????????????160Cys?Val?Ser?Cys?Ser?Asn?Cys?Lys?Lys?Ser?Leu?Glu?Cys?Thr?Lys?Leu
165?????????????????170?????????????????175Cys?Leu?Pro?Gln?Ile?Glu?Asn?Val?Lys?Gly?Thr?Glu?Asp?Ser?Gly?Thr
180?????????????????185?????????????????190Thr?Ala?Gly?Ala?Gly?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu?Ala
195?????????????????200?????????????????205Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr?Ile
210?????????????????215?????????????????220Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val?Leu225?????????????????230?????????????????235?????????????????240Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe?Glu
245?????????????????250?????????????????255Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Asn?Pro?Val?Val?Ser
260?????????????????265?????????????????270Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Ser?Thr
275?????????????????280?????????????????285Thr?Asp?Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp?Pro
290????????????????295??????????????????300Arg?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu?Pro305?????????????????310?????????????????315?????????????????320Ser?Pro?Ser?Arg?Leu?Pro?Gly?Pro?Ser?Asp?Thr?Pro?Ile?Leu?Pro?Gln
325?????????????????330?????????????????335
(2) information of SEQ ID NO:9:
(ⅰ) sequence signature:
(A) length: six amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: peptide
(ⅹ ⅰ) sequence description: SEQ ID NO:9Ala Gly Ala Ala Pro Gly1 5
(2) information of SEQ ID NO:10:
(ⅰ) sequence signature:
(A) length: 4 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: peptide
(ⅹ ⅰ) sequence description: SEQ ID NO:10Ala Gly Ala Gly1
(2) information of SEQ ID NO:11:
(ⅰ) sequence signature:
(A) length: 30 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
(ⅹ ⅰ) sequence description: SEQ ID NO:11TTTTCTCGAG ATGGCTACAG GTAAGCGCCC
(2) information of SEQ ID NO:12:
(ⅰ) sequence signature:
(A) length: 39 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
(ⅹ ⅰ) sequence description: SEQ ID NO:12ACCTGGGGCA GCACCGGCAC AGGAGACACA CTCGTTTTC
(2) information of SEQ ID NO:13:
(ⅰ) sequence signature:
(A) length: 42 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
(ⅹ ⅰ) sequence description: SEQ ID NO:13TGTGCCGGTG CTGCCCCAGG TTGCCCAGAA TGCACGCTAC AG 42
(2) information of SEQ ID NO:14:
(ⅰ) sequence signature:
(A) length: 36 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
(ⅹ ⅰ) sequence description: SEQ ID NO:14TTTTGGATCC TTAAGATTTG TGATAATAAC AAGTAC 36
(2) information of SEQ ID NO:15:
(ⅰ) sequence signature:
(A) length: 39 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
(ⅹ ⅰ) sequence description: SEQ ID NO:15CCGTGGACCA GCACCAGCAC AGGAGACACA CTCGTTTTC 39
(2) information of SEQ ID NO:16:
(ⅰ) sequence signature:
(A) length: 36 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
(ⅹ ⅰ) sequence description: SEQIDNO:16TGTGCTGGTG CTGGTCCACG GTGCCGCCCC ATCAAT 36
(2) information of SEQ ID NO:17:
(ⅰ) sequence signature:
(A) length: 32 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
(ⅹ ⅰ) sequence description: SEQ ID NO:17TTTTGGATCC TTATTGTGGG AGGATCGGGG TG 32
(2) information of SEQ ID NO:18:
(ⅰ) sequence signature:
(A) length: 27 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
(ⅹ ⅰ) sequence description: SEQ ID NO:18TTTTAGATCT CTTCTTGCAC AGTGGAC 27
(2) information of SEQ ID NO:19:
(ⅰ) sequence signature:
(A) length: 21 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
(ⅹ ⅰ) sequence description: SEQ ID NO:19TGTGGTGCCT GAGTCCTCAG T 21
(2) information of SEQ ID NO:20:
(ⅰ) sequence signature:
(A) length: 41 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
(ⅹ ⅰ) sequence description: SEQ ID NO:20ACTGAGGACT CAGGCACCAC AGCCGGTGCT GCCCCAGGTT G 1
(2) information of SEQ ID NO:21:
(ⅰ) sequence signature:
(A) length: 29 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
(ⅹ ⅰ) sequence description: SEQ ID NO:21TTTTTCTAGA GAAGCAGCAG CAGCCCATG 29
(2) information of SEQ ID NO:22:
(ⅰ) sequence signature:
(A) length: 75 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
(ⅹ ⅰ) sequence description: SEQ ID NO:22TTTTCCACAG CCAGGGTGGC ATTGATGGGG CGGCACCGTG GACCAGCACC AGCTGTGGTG 60CCTGAGTCCT CAGTG 75

Claims (20)

1, a kind of hybrid protein, it contains the aminoacid sequence that forms a kind of dimeric two kinds of coexpressions, and every seed amino acid sequence contains:
(a) at least a a kind of homology aggressiveness acceptor, a kind of chain of allos aggressiveness acceptor, the fragment of a kind of part and their reservation ligand-receptor binding ability of being selected from; And
(b) a kind of subunit of heterodimer protein hormone, other the subunit that has perhaps kept this subunit and it form its fragment of the ability of heterodimer;
Sequence (a) and (b) be direct bonded or by a kind of peptide connector bonded wherein, and wherein the sequence (b) in the sequence of each said two kinds of coexpression can be assembled and formed a kind of dimer complex body.
2, according to the described hybrid protein of claim 1, wherein said sequence (a) is selected from TBP1, TBP2 or still contains their fragment of ligand binding region; The extracellular region territory of IFN α/beta receptor or IFN γ acceptor; Gonadotropin acceptor or its extracellular segment; Light chain of antibody or its fragment randomly link with corresponding heavy chain; Heavy chain of antibody or its fragment; The monoclonal antibody zone; And IL-6, IFN-β, TPO or its fragment.
3, according to the described hybrid protein of claim 1, wherein said sequence (b) is to be selected from hCG, FSH, LH, the subunit of TSH or statin and their fragment.
4, according to the described hybrid protein of claim 1, wherein sequence (a) is to be connected with the N-terminal of sequence (b).
5, according to the described hybrid protein of claim 1, wherein sequence (a) is to be connected with the C-terminal of sequence (b).
6, according to the described hybrid protein of claim 1, each of the aminoacid sequence of wherein said two kinds of coexpressions comprises the sequence of TBP1 or its fragment, this fragment is corresponding to amino-acid residue 20-161 or the 20-190 of TBP1, as sequence (a), and the α of hCG separately and β subunit or its fragment, as sequence (b).
7, according to the described hybrid protein of claim 1, each of the aminoacid sequence of wherein said two kinds of coexpressions comprises the extracellular region territory of gonadotropin acceptor, as sequence (a), and the α of gonadotropin separately and β subunit, as sequence (b).
8, according to the described hybrid protein of claim 7, wherein said sequence (a) is that fsh receptor extracellular region territory and sequence (b) are the FSH subunits.
9, according to the described hybrid protein of claim 7, wherein said sequence (a) is connected by a peptide connector with (b).
10, according to the described hybrid protein of claim 9, wherein said peptide connector has a kind of restriction enzyme site.
11, according to the described hybrid protein of claim 10, wherein said restriction enzyme site is a kind of zymoplasm cleavage site.
12, according to the described hybrid protein of claim 10, wherein said restriction enzyme site is by a kind of enzyme identification and cutting that is found in the ovary.
13, according to the described hybrid protein of claim 9, wherein said peptide connector serves as a kind of flexible hinge.
14,, wherein between two subunits (b), one or more covalent linkage have been added according to the described hybrid protein of claim 1.
15, a kind of dna molecular of the described hybrid protein of claim 1 of encoding.
16, the expression vector that contains the described dna molecular of claim 15.
17, a kind of host cell, it contains the described expression vector of claim 16 and can express said impurity protein.
18, a kind of method of producing hybrid protein comprises described host cell of cultivation claim 17 and the recovery hybrid protein by its expression.
19, a kind of pharmaceutical composition, it contains the described hybrid protein of claim 1 and pharmaceutically acceptable carrier and/or vehicle.
20, a kind of sophisticated method of folliculus of inducing comprises that the pharmaceutical composition that will contain the described hybrid protein of claim 8 is administered to the object of being executed of these needs.
CNB971924112A 1996-02-20 1997-02-20 Hybrid proteins which form heterodimers Expired - Fee Related CN1261579C (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US1193696P 1996-02-20 1996-02-20
US60/011,936 1996-02-20

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Publication Number Publication Date
CN1212017A true CN1212017A (en) 1999-03-24
CN1261579C CN1261579C (en) 2006-06-28

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EP (1) EP0894141B1 (en)
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KR (1) KR100369985B1 (en)
CN (1) CN1261579C (en)
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AU (1) AU706504B2 (en)
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CZ (1) CZ291212B6 (en)
DE (1) DE69733309T2 (en)
DK (1) DK0894141T3 (en)
EA (1) EA002474B1 (en)
EE (1) EE04025B1 (en)
ES (1) ES2241040T3 (en)
HU (1) HUP9900619A3 (en)
IL (1) IL125864A (en)
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CN108129573B (en) * 2007-09-21 2021-10-08 加利福尼亚大学董事会 Targeted interferons exhibit potent apoptotic and antitumor activity
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